Mitochondrial superoxide (O2*-) production is an important mediator of oxidative cellular injury. Succinate-cytochrome c reductase (SCR) of the electron transport chain has been implicated as an essential part of the mediation of O2*- generation and an alternative target of nitric oxide (NO) in the regulation of mitochondrial respiration. The Q cycle mechanism plays a central role in controlling both events. In the present work, O2*- generation by SCR was measured with the EPR spin-trapping technique using DEPMPO (5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide) as the spin trap. In the presence of succinate, O2*- generation from SCR was detected as the spin adduct DEPMPO/*OOH. Inhibitors of the Q(o*-) site only marginally reduced (20-30%) this O2*- production, suggesting a secondary role of Q(o*-) in the mediation of O2*- generation. Addition of cyanide significantly decreased (approximately 70%) O2*- production, indicating the involvement of the heme component. UV-visible spectral analysis revealed that oxidation of ferrocytochrome b was accompanied by cytochrome c(1) reduction, and the reaction was mediated by the formation of an O2*- intermediate, indicating a direct role for cytochrome b in O2*- generation. In the presence of NO, DEPMPO/*OOH production was progressively diminished, implying that NO interacted with SCR or trapped the O2*-. The consumption of NO by SCR was investigated by electrochemical detection using an NO electrode. In the presence of succinate, SCR-mediated NO consumption was observed and inhibited by the addition of superoxide dismutase, suggesting the involvement of O2*-. Under the conditions of argon saturation, the NO consumption rate was not enhanced by succinate, suggesting a direct role for O2*- in the mediation of NO consumption. In the presence of succinate, oxidation of the ferrocytochrome b moiety of SCR was accelerated by the addition of NO, and was inhibited by argon saturation, indicating an indirect role for cytochrome b in the mediation of NO consumption.